Technical Field
The present invention relates generally to methods and apparatuses for processing samples using thin films. More specifically, the invention is related to methods and apparatuses for providing adjustable volume accommodation to process samples.
Description of the Related Art
A wide variety of techniques have been developed to prepare and analyze biological samples. Example techniques include microscopy, micro-array analyses (e.g., protein and nucleic acid micro-array analyses), and mass spectrometric methods. Samples are prepared for analysis by applying one or more liquids to the samples. If a sample is treated with multiple liquids, both the application and subsequent removal of each of the liquids can be important for producing samples suitable for analysis.
Microscope slides bearing biological samples, e.g., tissue sections or cells, are often treated with one or more dyes or reagents to add color and contrast to otherwise transparent or invisible cells or cell components. Samples can be prepared for analysis by manually immersing sample-bearing slides in containers of dyes or other reagents. This labor intensive process often results in inconsistent processing and carryover of liquids between containers. Carryover of liquids leads to contamination and degradation of the processing liquids. These types of manual processes often utilize excessive volumes of liquids resulting in relatively high processing costs, especially if the dyes or other reagents are expensive and are prone to degradation due to carryover.
“Dip and dunk” automated machines immerse samples in liquids similar to manual immersing techniques. These automated machines can process samples in batches by submerging racks carrying microscope slides in open baths. Unfortunately, relatively large amounts of reagents are in bath containers of the dip and dunk automated machines. Similar to manual processes, if the liquids are expensive reagents, processing costs may be relatively high, especially if significant amounts of reagents are wasted. Reagent bath containers may be frequently emptied because of contamination due to carryover. Open containers are also prone to evaporative losses that may significantly alter the concentration of the reagents resulting in inconsistent processing. It may be difficult to process samples without producing significant volumes of waste that may require special handling and disposal.
Immunohistochemical and in situ hybridization staining processes are often used to prepare specimens. The rate of immunohistochemical and in situ hybridization staining of sectioned fixed tissue on a microscope slide is limited by the speed at which molecules (e.g., conjugating biomolecules) can diffuse into the fixed tissue from an aqueous solution placed in direct contact with the tissue section. Tissue is often “fixed” immediately after excision by placing it in a 10% solution of formaldehyde, which preserves the tissue from autocatalytic destruction by cross-linking much of the protein via methylene bridges. This cross-linked tissue may present many additional barriers to diffusion, including the lipid bilayer membranes that enclose individual cells and organelles. Conjugate biomolecules (antibody or DNA probe molecules) can be relatively large, ranging in size from a few kilo Daltons to several hundred kiloDaltons, which constrains them to diffuse slowly into solid tissue with typical times for sufficient diffusion being in the range of several minutes to a few hours. Typical incubation conditions are thirty minutes at 37 degrees centigrade.
The diffusion rate is often driven by a concentration gradient so the diffusion rate can be increased by increasing the concentration of the conjugate in the reagent. Unfortunately, conjugates are often very expensive, so increasing their concentration is wasteful and often not economically viable. Additionally, the excessive amount of conjugate that is driven into the tissue, when high concentrations are used, is entrapped in the tissue, and is difficult to rinse out and causes high levels of non-specific background staining. In order to reduce the noise due to non-specific background staining and increase the signal of specific staining, low concentrations of conjugate with long incubation times are often used to allow the conjugate to bind only to the specific sites.
Conventional histology staining instruments often use relatively large volumes of reagent (100 μl) in a puddle of typically 300 μl of buffer. This produces a rather low concentration of the reagent in the puddle that resides over the tissue. Some conventional instruments mix the reagent by alternating tangential air jets onto an overlaying oil layer that rotates and counter-rotates when contacted by the alternating air jets, thereby imparting motion into the underlying aqueous puddle. This mixing is slow and not particularly vigorous and creates significant evaporation losses. Large volumes of rinse liquid are used to physically displace the large puddles of low concentration reagents which are covered with oil. This rinsing procedure produces large volumes of waste liquid which may be hazardous waste, and can physically disrupt the tissue by the vigorous washing action.